The locations and patterns of methylated residues in DNA play an important role in many areas of biological research, not least in epigenetics and oncology. Epigenetic patterns of modification control the structure of chromatin, and thereby regulate gene expression. Methylation of DNA at specific sites can prevent the binding of transcription factors (a direct effect), or groups of methylated sites can attract methyl-binding domain (MBD) proteins, which act in concert with histone deacetylases to alter chromatin structure and silence gene expression (an indirect effect). Regions in mammalian genomes rich in CG dinucleotides, called CpG islands, are often indicative of methylation resistance and active gene expression. Abnormal methylation patterns have been associated with cancer due to resultant changes in gene expression. Large-scale investment in the “Human Epigenome Project” (HEP), an effort to map sites of cytosine methylation in the human genome, attests to the growing importance of knowledge of patterns and sites of DNA methylation.
Several technologies for analyzing methylated DNA are available at present. These include analyzing overall methylation content of a DNA sample (e.g., a genome), the degree of methylation at a particular site in a sample, and the pattern of methylation of multiple sites in cis on a DNA strand in a particular region, or on profiles of methylation at selected sites throughout the genome. The technologies to achieve the above largely depend on restriction digestion or bisulfite conversion (Laird, P. W. Nat Rev Cancer 3, 253-266 (2003)).
Conventional restriction digestion relies on the fact that methylation at or near restriction endonuclease sites can block cleavage by those nucleases. Differing methylation patterns, therefore, will cause differences in the patterns of cleavage by various restriction enzymes. One of the oldest techniques for examining the products of these restriction digests is the Southern blot. This process is relatively time-consuming, however, and requires a large amount of DNA for analysis. Furthermore, determination of precise methylation sites is dependent on a priori knowledge of the sequence being examined unless an appropriate unmethylated control sample is available. In addition, “Lack of cleavage” is a less desirable method of detection than provided by modified cytosine restriction A (McrA) because inactive enzyme or incomplete digestion can lead to a false positive result.
Treatment of DNA with sodium bisulfite converts unmethylated, but not methylated, cytosine to uracil over short timespans. Many techniques have been developed to exploit this phenomenon, but by far the most common bisulfite-based methods involve amplification of converted DNA by PCR and sequencing. While this method allows for the precise identification of methylation sites, it precludes the analysis of a large number of noncontiguous methylation sites simultaneously. Identifiable sites in a given experimental reaction must be within a single PCR amplicon, or at best a moderate number of amplicons afforded by multiplex PCR.